5.3 Results and discussion
5.3.2 Growth inhibition studies
The cytotoxicity of lipoplexes was assessed under the conditions adopted for transfection studies, as there often exists a correlation between the effect of vectors on cell growth and their gene transfer potential (Wiethoff and Middaugh, 2003). Throughout the study lipoplexes were assembled in HBS to reduce the possibility of the assays being influenced by osmotic effects (Singh et al., 2007). This buffer maintains osmolarity at approximately 290 mosmol/kg; and is optimal for cultured human cells as it corresponds to that of human plasma (Freshney, 2005).
In this study, cytotoxicity profiles of the liposomes formulated are represented, in Figures 5.7 and 5.8, as the percentage cell survival after exposure to lipoplexes assembled at different Chol-T:DNA ratios by weight. A prevalent trend in the literature is that cytotoxicity increases with increasing lipoplex charge ratio (Lv et al., 2006; Masotti et al., 2008). This is largely because a higher charge ratio correlates with the inclusion of higher levels of cationic amphiphiles, which is in turn associated with adverse effects on a molecular level (Lasic, 1997). However, Masotti and colleagues (2008) have reported that this pattern does not hold true for all cationic liposome formulations. This is possibly a consequence of helper lipids included in their composition, as these also influence cell growth; and the specific cell line employed in the investigation. Moreover, the lipid concentration to which cells are exposed, the duration of exposure and the cell density employed in in vitro growth inhibition studies are known to influence the end result (Pazner and Jansons, 1979). Therefore, in comparing the cellular response to each liposomal carrier, at the optimal, sub- and super-optimal DNA- binding ratios, due consideration was given to the cytofectin and total lipid concentration (Table 5.1) introduced per cell sample.
131
Chol-T:DNA (w/w) ratio
% cell survival
0
1.5:1
1.75:1
2.0:1 0
50 100 150
200 HepG2
HEK293
Chol-T:DNA (w/w) ratio
% cell survival
0
0.75:1
1.0:1
1.25:1 0
50 100 150
200 HepG2
HEK293
Fig. 5.7 a)
**
***
#
***
b)
132
Chol-T:DNA (w/w) ratio
% cell survival
0
0.5:1
0.75:1
1.0:1 0
50 100 150
200 HepG2
HEK293
Chol-T:DNA (w/w) ratio
% cell survival
0
1.0:1
1.25:1
1.5:1 0
50 100 150
200 HepG2
HEK293
Figure 5.7: Growth inhibition studies of non-pegylated liposomes, a) 1; b) 2; c) 3; and d) 4.
Cells in serum-free medium were subjected to 4 hour exposure to lipoplexes (10 µl in HBS) prepared from 0.5 µg pCMV-luc DNA and different amounts of liposome corresponding to optimal, sub- and super-optimal DNA-binding ratios. Each column represents the mean ± SD (n = 3). **P < 0.01, ***P < 0.001 vs. HepG2; #P < 0.05 vs. the relevant control.
c)
d) #
133
Chol-T:DNA (w/w) ratio
% cell survival
0
3.0:1
3.5:1
4.0:1 0
50 100 150
200 HepG2
HEK293
Chol-T:DNA (w/w) ratio
% cell survival
0
3.0:1
3.5:1
4.0:1 0
50 100 150
200 HepG2
HEK293
Fig. 5.8 a)
*
b)
134
Chol-T:DNA (w/w) ratio
% cell survival
0
3.0:1
3.5:1
4.0:1 0
50 100 150
200 HepG2
HEK293
Chol-T:DNA (w/w) ratio
% cell survival
0
2.5:1
3.0:1
3.5:1 0
50 100 150
200 HepG2
HEK293
Figure 5.8: Growth inhibition studies of pegylated liposomes, a) 5; b) 6; c) 7; and (d) 8. Cells in serum-free medium were subjected to 4 hour exposure to lipoplexes (10 µl in HBS)
prepared from 0.5 µg pCMV-luc DNA and different amounts of liposome corresponding to optimal, sub- and super-optimal DNA-binding ratios. Each column represents the mean ± SD (n = 3). *P < 0.05 vs. HepG2.
c)
d)
135 In general, pegylated and non-pegylated lipoplexes demonstrated favourable biocompatibility towards both HepG2 and HEK293 cells. However, cellular growth patterns in response to several liposome formulations differed noticeably in the two cell lines. For example, survival of hepatoma cells increased with increasing concentrations of liposome 1 within a range of 1.8 – 2.4 µg/well, having promoted growth by approximately 33 % at the super-optimal DNA-binding ratio. In contrast, the viability of the kidney cell line steadily decreased to 60 %, after an initial increase in cell numbers, in response to the same concentrations of the Chol-T/DOPE formulation (Figure 5.7a). Other authors have also reported that equimolar combination of Chol-T and DOPE correlates with cell survival rates in excess of 60 %
(Balram et al., 2009; Singh and Ariatti, 2003; Singh et al., 2010). Furthermore, increasing the cytofectin:DNA ratio of lipoplexes assembled from pegylated preparations 5 – 8 in HepG2 cells was, in general, accompanied by a measurable change in cell numbers. However, in HEK293 cells, a similar response to the three different Chol-T:DNA ratios was observed with the exception of liposome 6 (Figure 5.8a, c, and d). In instances where similar effects on cell growth were observed between cell lines, such as the response to liposome 4, (Figure 5.7d), the growth inhibitory or growth stimulatory effect of individual lipoplexes often appeared more profound in one cell line than the other. Across the eight vector formulations, maximal growth inhibition was recorded at 50 % with respect to hepatocytes upon exposure to the Chol-T/DOPE/SH02 lipoplexes at the ratio of 1.25:1; and at 40 % in HEK293 cells in the presence of Chol-T/DOPE lipoplexes at a ratio of 2:1. These findings are not unusual as cell- specific responses to several cationic liposomal carriers have been documented (Lv et al., 2006; Ma et al., 2007; Romøren et al., 2004). According to Romøren and coworkers (2004) this may be attributed to differences in cellular accumulation of the vector, as a consequence of variation in cell surface characteristics; and its intracellular processing among cell lines.
A comparison of the cytotoxicity profiles of each liposome in a single cell line demonstrates that, in addition to lipid concentration, the type, properties and relative proportions of individual lipid components greatly influences the effect of transfecting complexes on cell proliferation. Similar statements were forwarded by Lv and coworkers (2006) in a review of cationic lipid-mediated toxicity. As an illustration, the introduction of the galactosylated lipid SH02 into the Chol-T/DOPE formulation, reversed the cell proliferation pattern that was observed upon exposure of HepG2 cells to liposome 1, with increasing Chol-T:DNA ratio.
Figure 5.7b, shows that survival of hepatoma cells steadily decreased until maximal growth
136 inhibition of 50 % was attained at the super-optimal (1.25:1) DNA-binding ratio.
Transfection studies support the fact that these galactose-modified lipoplexes, were effectively internalised by HepG2 cells, due to ASGP-R-mediated endocytosis (refer to 5.3.3). The cell-specific accumulation mediated by liposome 2, may account for its more profound cytotoxic effect, especially at higher liposome and cytofectin concentrations. As a further example, modification of the Chol-T/DOPE formulation with the imidazolylated lipid, SH04, at the same molar ratio as SH02, led to improved cell tolerance, having permitted cell survival in excess of 84 % at all ratios investigated (Figure 5.7c). Due to the stronger DNA- binding affinity of liposome 3 (Chol-T/DOPE/SH04), smaller amounts of liposome, corresponding to lower levels of cytofectin, were required to prepare lipoplexes from this formulation, than liposomes 1 and 2. In this regard, it has been reported that cationic liposome formulations that are able to complex DNA with minimal lipid content, are favourable gene transfer agents as these generally display minimal cytotoxicity (Muñoz- Úbeda et al., 2011).
Among the non-pegylated carriers, lipoplexes prepared from liposomes 3 and 4, gave the most favourable cytotoxicity profiles in both cell lines, with cell survival in excess of 84 % at the different DNA-binding ratios investigated. In addition, their pegylated counterparts were best tolerated among the sterically stabilised preparations: liposome 8 resulted in a slight inhibition of growth, of approximately 10 %, when complexed with DNA at ratios of 2.5:1 and 3:1 in HepG2 cells alone (Figure 5.8d); while liposome 7 mediated a slight increase in the growth of both cell lines at all DNA-binding ratios investigated (Figure 5.8c). On the whole, pegylated lipoplexes prepared at the optimal, sub- and super-optimal DNA-binding ratios contained larger amounts of liposome and, accordingly, cytofectin, than their non- pegylated counterparts due to their weaker DNA-binding capacity. Nonetheless, the pegylated liposomes permitted cell survival in excess of 66 %, and often elicited growth promoting effects (Figures 5.8a-d). In fact, other authors have reported that modification of cationic vectors with PEG enhanced the biocompatibility of the carriers (Nagasaki et al., 2004; Narainpersad et al., 2012). Although this study was limited to in vitro assays, it is worthy of note that steric stabilisation of liposomes using DSPE-PEG2000 at maximal concentration of 10 %, was shown to minimise liposome-induced inflammatory toxicity in animal models, by preventing interactions between liposomes and cells of the immune system (Filion and Phillips, 1998; Sakurai et al., 2002; Zhang et al., 2005).
137